Mycobacterium tuberculosis is a major opportunistic pathogen in patients with HIV/AIDS. Current tuberculosis treatment regimes are severely hampered by the occurrence of multidrug resistant strains of M. tuberculosis (MDR-TB) and there is a critical need for the development of novel chemotherapeutics. We will design and synthesize inhibitors of InhA, the enoyl-reductase enzyme from M. tuberculosis (MTB). InhA, which catalyzes the final step in the fatty acid elongation pathway, is a target for the activated form of isoniazid (INH) a front-line anti-TB drug. Importantly, the large majority of INH-resistant isolates result from mutations in KatG, the enzyme that activates INH, rather than from mutations in the ultimate drug target(s). Consequently, it is hypothesized that inhibitors of InhA, which circumvent the need for KatG activation, will form an effective basis for tackling MDR-TB. The lead compound for inhibitor discovery is triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol), a compound that inhibits InhA with a Ki value of 0.22 mu/M and which has an MIC99 for MTB of 5 mu/g/mL (17 mu/M). The proposed research includes the following specific aims: (1) We will synthesize a library of triclosan analogs using directed synthesis and parallel synthetic methods. (2) We will determine the inhibitory potency of the analogs and analyze the interaction of promising compounds with InhA using techniques such as kinetics, X-ray crystallography and Raman spectroscopy. (3) We will determine MIC99 values against sensitive and resistant TB strains. To evaluate whether InhA is the target for these compounds we will (i) determine MIC99 values in an MTB strain overexpressing InhA and (ii) use promoter-specific reporter fusions coupled with radiolabeled uptake studies and photoaffinity labeling to investigate the mechanism of compound action in live bacteria. The in vivo activity of promising InhA inhibitors will be further analyzed through the NIAID-funded Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF). Information derived from Aims 2-3 will direct the synthesis of additional compounds which will then be submitted for further rounds of screening and evaluation.